Methylmercury (MeHg) poisonings are well documented chemical disasters in human history. The case for significant MeHg toxicity to the central nervous system (CNS) is strongly supported by both in vivo and in vitro studies. Only recently have astrocytes been found to preferentially accumulate MeHg and their responses considered in the etiology of MeHg-induced neuronal toxicity. Exposure to MeHg results in astrocytic swelling with concomitant inhibition of glutamate uptake, changes in regulatory volume decrease, and increased release of excitatory amino acids (EAAs), such as glutamate. The primary working hypothesis of this collaborative proposal postulates that the neurotoxic effect of MeHg is associated with elevated concentrations of extracellular glutamate, in turn, leading to activation of neuronal N-methyl-D- aspartate receptors. Mechanisms invoked for the increased concentration of extracellular glutamate include altered enzymatic activities of glutamine synthetase and glutaminase, as well as the levels of these enzymes and glutamate transporters. Our initial approach will e apply state-of-the-art molecular biology approaches to isolate glutamine synthetase and glutaminase, and study their activities in acutely-, chronically-, and in situ-MeHg exposed astrocytes. Levels of glutamine synthetase, glutaminase and the glutamate transporters will be determined by Western blotting technique. Expression of these genes both in controls and MeHg-treated cells will be correlated to the availability of the respective messenger RNA, quantified by means of RNA-excess solution hybridization method. We will also study possible mutations of the genes coding for glutamine synthetase, glutaminase and the glutamate transporters by means of PCR-SSCP (PCR-directed single strand conformational polymorphism) and dideoxy DNA sequencing in controls and MeHg exposed astrocytes. The overall long term objective of this collaborative research is to continue to elucidate the mechanisms of MeHg neurotoxicity and the potential deleterious effects of astrocytic failure to control glutamate homeostasis in its etiology.